Some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) using a lactate dehydrogenase (Ldh) in concert with the electron-transferring proteins EtfA and EtfB. The electron cryo-microscopically characterized (Ldh-EtfAB)2 complex of Acetobacterium woodii at 2.43 Å resolution consists of a mobile EtfAB shuttle domain located between the rigid central Ldh and the peripheral EtfAB base units. The FADs of Ldh and the EtfAB shuttle domain contact each other thereby forming the D (dehydrogenation-connected) state. The intermediary Glu37 and Glu139 may harmonize the redox potentials between the FADs and the pyruvate/lactate pair crucial for FBEC. By integrating Alphafold2 calculations a plausible novel B (bifurcation-connected) state was obtained allowing electron transfer between the EtfAB base and shuttle FADs. Kinetic analysis of enzyme variants suggests a correlation between NAD+ binding site and D-to-B-state transition implicating a 75° rotation of the EtfAB shuttle domain. The FBEC inactivity when truncating the ferredoxin domain of EtfA substantiates its role as redox relay. Lactate oxidation in Ldh is assisted by the catalytic base His423 and a metal center. On this basis, a comprehensive catalytic mechanism of the FBEC process was proposed.Lactate oxidation with NAD In the confurcation mode H2 is produced from reduced ferredoxin (Fdred) and NAD(P)H (– state (– (< –700 mV) by Fdred (E ≈ –500 mV) is strongly coupled with the exergonic reduction of FAD•– to FADH– (E >+200 mV) by an electron originating from the high-potential (weak) electron donor (E≤0 mV) (–) = –911 mV and E(FAD•–/FADH–) = +359 mV are experimentally determined for the bifurcating NADPH dependent Fdox:NAD+ oxidoreductase  embedded into a modular, mostly soluble enzyme complex . In the eduction . This tweduction  that oxi NAD(P)H . As key (– state . In the (E≤0 mV) . One-eleeductase . Strongl complex . The FBE complex .+ as electron acceptor driven by simultaneous co-oxidation of Fdred (via glycolysis to the endproduct acetate (The electron-confurcating lactate dehydrogenase/electron-transferring flavoprotein (Ldh-EtfAB) complex, the research subject of this report, catalyzes the endergonic oxidation of lactate to the central cellular metabolite pyruvate  with NADof Fdred . This FBof Fdred . Lactate acetate .The confurcating Ldh-EtfAB complex belongs to the bEtf family, the largest and most diverse group of FBEB/FBEC enzymes . bEtf fa17CxxCxxC) Ldh-EtfAB possesses a Fd domain with one [4Fe-4S] cluster  structure complemented by kinetic analysis of various enzyme variants.lctBCD genes from A. woodii were cloned into the expression vector pET21a and expressed in the Escherichia coli strain BL21(DE3)ΔiscR ΔiscR . The LdhE3)ΔiscR  that matE3)ΔiscR  and ≈250E3)ΔiscR .A. woodii suberate (BS3), because multiple attempts without cross-linking failed. From 9,788 micrographs collected and 674,283 particles extracted a twofold-averaged density map at a mean resolution of 2.43 Å was calculated  state  state , in whicd) state . In bifud) state  by applyd) state  and aligd) state . The Etfd) state  that devd) state .The oscillation of the EtfAB shuttle domain between the D- and B-states is realized by a fixed interface between the rigid Ldh core and EtfAB base platforms and a variable interface either between the EtfAB shuttle domain and Ldh or between the EtfAB shuttle domain and the EtfAB base . The fixA. woodii are 2.6 Å , 2.6 Å , 3.5 Å  and 2.4 Å . Except for the membrane-associated Ldh CMH-type flavoenzymes are homodimers. D-FAD is embedded between the two α + β subdomains except for the isoalloxazine ring that protrudes beyond the FAD domain towards the cap domain. Therefore, the isoalloxazine is well accessible from bulk solvent  subdivided into two α + β subdomains, a cap domain (219-417) characterized by an antiparallel β-sheet element and an extended C-terminal arm (418-467) attached to the FAD domain . This fo solvent .si-side the substrate binding site can be easily reached by lactate/pyruvate via a wide gate  members  reflecte members , CarCDE  members , and Fix members . The cormbers 6 oxidoreductase activities were 50% lower than those of the wild-type enzyme indicating that these mutations primarily do not affect the individual redox reactions but their coupling. In the context of previous data 6 oxidoreductase activity 13.9±5.4 and 14.6±4.8 U/mg, respectively (Clostridium propionicum (Cprop2325) highly related to their bifurcating partners contain an insertion region that may block Fd binding, as already predicted  was truncated or cysteines 41, 44, and 47 coordinating the [4Fe-4S] clusters were exchanged to alanine. In these [∆Fe/S-domain] and [∆Fe/S] variants, the irons are completely lost (0.3±0.2 and 0.5±0.3 mol Fe/mol protein); the stability and subunit composition were, however, not affected  and the ectively . In contectively . In summredicted , in a mavia a hydride transfer to N5 forming d-FADH− (− rapidly flows to a-FAD thereby generating d-FADH• (d-FAD•−) and a-FAD•− (a-FADH•) (−. (3) NAD+ binds into a pocket at the si-side of b-FAD as verified by site-directed mutagenesis experiments  External Fd binds to the mobile Fd domain of EtfA, thereby rigidifies the latter and adjusts a productive ET geometry between their two [4Fe-4S] clusters and b-FAD  Upon release of NADH and/or Fdox the EtfAB shuttle domain swings back into the D-state and a-FAD uptakes the remaining electron of d-FADH• (d-FAD•−). In analogy to the first round b-FAD is reduced in the B-state by one-electron transfers from Fdred and a-FAD•− rotated before from the Ldh to the EtfAB surface  . It is weriments  in agreeeriments . In the  binding . Accordi binding  and supp binding  NAD+ binnd b-FAD . A relatnd b-FAD . (5) Per B-state . (6) Fdrng b-FAD  and 9A.  B-state . Accordi B-state . (8) The surface . Finally to NADH .+ and ferredoxin, solely the high-potential substrate varies and determines the direction of the reaction. As mentioned, the redox potential E°’ of the pyruvate/lactate pair is with –190 mV substantially lower than that of the crotonyl-CoA/butyryl-CoA pair of –10 mV, which turns a bifurcation into a confurcation event  were amplified from chromosomal DNA using the primers lctBCD_pET21a_for and lctBCD_pET21a_rev (pET21a (5406 bp) was amplified using the primers pet21a_for and pet21a_rev (lctBCD and pET21a were fused via Gibson Assembly (New England Biolabs) and transformed into E. coli HB101 (lctC coding for EtfA by using corresponding primers (pET21a_lctBC-StrepD was the template for site-directed mutagenesis. Nucleic acid changes were introduced using corresponding primers at desired loci in the encoding genes (pET21a plasmids were transformed into E. coli BL21(DE3) ∆iscR.The genes T21a_rev . The expt21a_rev . Afterwali HB101 . A seque primers . The resng genes . GeneratE. coli BL21(DE3)∆iscR was grown aerobically in modified LB-medium  supplemented with 4 mM ammonium-iron(II)citrate and 25 mM glucose at 37°C. At an OD600 of 0.5–0.8 the culture was transferred into a sterile, anaerobic Müller-Krempel flask  and supplemented with 2 mM cysteine and 20 mM fumarate. After closing the culture with a butyl stopper, the flask was further incubated at 16°C. As soon as the culture was cooled down to 16°C gene expression was induced by addition of IPTG to a final concentration of 1 mM. All steps from this point onward were executed in an anaerobic chamber  with a mixed gas phase of N2/H2 (95:5 [v/v]). After 16–19 hr cells were anaerobically harvested, resuspended in 200 mL buffer W  glycerol, 4 µM resazurin, 5 µM FAD, 2 mM DTE, pH 8) and disrupted once in a French press  after addition of DNase I and 0.5 mM PMSF . The lysate was centrifuged  to separate undisrupted cells from the crude extract. Subsequently, the Ldh-EtfAB complex was purified by affinity chromatography on Strep-Tactin high-capacity material  using the elution buffer E (buffer W+5 mM desthiobiotin). Fractions containing Ldh-EtfAB were pooled and concentrated via ultrafiltration  to a volume of 500 µL. The concentrated sample was separated with a flow rate of 0.5 mL/min on a Superdex 200 10/300 GL column , previously equilibrated with buffer W. For cryo-EM studies buffer S  was used for the size exclusion step. Afterwards, the Ldh-EtfAB complex was stabilized with the bis(sulfosuccinimidyl)suberate (BS3) crosslinker  by incubating the sample (≈ 1 mg) with 1 mM BS3 for 20 min at room temperature. The assay was stopped by addition of 50 mM Tris. To get rid of impurities, the crosslinked-sample was again separated with a flow rate of 0.5 mL/min on a Superdex 200 10/300 GL column , previously equilibrated with buffer S.2 atmosphere at 30°C in 1.8 mL anaerobic cuvettes (Glasgerätebau Ochs), which were sealed with rubber stoppers. Physiological Fdred-dependent lactate:NAD+- and pyruvate-dependent NADH:Fdox oxidoreductase activities are measured as previously described (3Fe(CN)6- and NADH:DCPIP oxidoreductase activities using 1 mM K3Fe(CN)6 (ferricyanide) and 500 µM DCPIP (dichlorophenol indophenol), respectively. The NAD+/NADH (ε=6.3 mM–1 cm–1), Fd (ε=13.1 mM–1 cm–1) purified from C. pasteurianum  or K3Fe(CN)6 (ε=1 mM–1 cm–1) oxidations/reductions were monitored at 340, 430, 600, or 420 nm by UV/Vis spectroscopy, respectively. One unit (U) equals 2 μmol of electrons transferred per min.Kinetic measurements were routinely performed in a Nescribed . The sameurianum , DCPIP  were glow discharged thrice with a PELCO easiGlow device at 15 mA for 45 s. Four µl of protein with a concentration of 1.8 mg/ml were applied to a freshly glow-charged grid, blotted for seconds at 4 °C, 100% relative humidity and a blot force of +20. Grids were vitrified by plunging into liquid ethane using a Vitrobot Mark IV device (Thermo Scientific).Movies were recorded using a Titan Krios G3i microscope operated at 300 kV (Thermo Scientific) and equipped with a Gatan BioQuantum imaging filter using a 30 eV slit width and a K3 direct electron detector. Data were collected at a nominal magnification of 105,000×in electron counting mode using aberration-free image-shift (AFIS) correction in EPU (Thermo Scientific). Applied parameters are listed in via 2D classification in RELION-3.1. Unsupervised Initial model building, 3D classification, 3D refinement, along with post-processing steps like CTF refinement, Bayesian polishing and final map reconstructions were performed with RELION-3.1. Maps were visualized with Chimera  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Pimchai Chaiyen as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Philip Cole as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Andrea Mattevi (Reviewer #3).Thank you for submitting your article "Structure-based electron-confurcation mechanism of the Ldh-EtfAB complex" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.+ to NADH. Because electrons are being supplied by the dehydrogenase, the ETF executes confurcation in contrast to all of those elucidated so far, which function in the opposite direction to effect bifurcation. As electron-confurcation and electron-bifurcation have emerged as important paradigms of cellular bioenergetics, the data reported herein pave a way for future exploration of similar electron transfer systems. The findings reported here can lay the ground for understanding structural biology related to these intriguing bioenergetic systems and may possibly be mentioned in a general textbook in the future.This paper describes a high-resolution cryoEM structure of the lactate dehydrogenase-electron transferring flavoprotein complex that provides critical insight into the basic metabolic pathway of anaerobic fermentation. What is novel is that this is the first structure of a complex wherein the dehydrogenase runs as such, rather than as a reductase. The lactate produced by glycolysis is oxidized to pyruvate with concomitant reduction of NADGeneral clarifications:The manuscript needs considerable improvement with an eye to the readers who are not familiar with the electron bifurcation and other known electron bifurcating complexes.The term shuttle is used to mean one domain of the ETF. Please provide a definition at the time of first use, so that this is clear. Specific comments:1. It is interesting to see the impact that alphafold can have on experimental structure analysis. However, as a general comment, the authors tend to use the alphafold structures almost uncritically, as if they were experimentally determined. The manuscript should refer to the quality indicators given by alphafold and indicate how reliable the models are in particular around the regions that are mechanistically discussed in the text.2. The last paragraph of the main text (lines 446-460) should be mostly re-written and clarified. The sentence "The midpoint redox potential of the pyruvate/lactate pair is -190 mV, which is substantially lower than that of the crotonyl-CoA/butyryl-CoA pair of 10 mV, which turns a bifurcation into a confurcation event" requires a better explanation as, here, we are at the heart of the functional and structural principles governing the LDH complex. Figure 7B remains thereby difficult to be fully understood. Likewise, the sentence "When assuming that the redox potentials of the electron carriers between b-FAD and lactate also differ by ca. 180 mV the microenvironment of a-FAD and d-FAD should be distinguished between Bcd-EtfAB and Ldh-EtfAB ." remains honestly quite obscure to me. Likewise, the text should improve the discussion about the role of Gly37 and Gly139 and why the flavin of LDH should feature a lower redox potential compared to the analogous flavin of Bcd.3. The manuscript should explain more clearly and from the beginning, the difference between confurcation and bifurcation. Likewise, the introduction should be accompanied by a scheme depicting the overall mechanism of electron bifurcation/confurcation in general and with reference to the reaction by the LDH system.4. The abstract should be improved. Specifically, the text at lines 38-42 will be difficult to follow, at least for the casual reader.5. Figure 2 legend, explain the meaning of CODH.6. Line 189, the meaning of "promiscuous" EtfAB should be described explicitly.7. Line 306, please describe where the residues 122-130 are located in the structure of the complex and why they define an electron-bifurcating system. Why are these residues making the difference between bifurcating and non-bifurcating enzymes?8. The supplement figures of Table 1 should probably be moved to the main text as they are crucial for understanding the rationale of the mutagenesis experiments.9. Line 322, for the analysis of the catalytic role of Arg205, the authors may refer to recently published work (doi.org/10.1016/j.jbc.2022.101733).10. Line 180 and throughout, CarCDE and Fix-EtfABX should be described in more detail as the authors often refer to them for their structural and functional comparisons.11. Line 437, can the authors comment on the proton sources that couple protonation and reduction of the flavins.12. Figure 7, the legend states that the two-electron reduced FAD is shown in orange but the drawing shows it colored in red.13. Figure 7 legend, what is an "escapement" mechanism? Explain.14. The figure supplement 1 of Figure 4 does not show the residues listed in the figure legend .15. Line 102, rephrase, for example replacing 'holded' by 'held'. 16. Line 125: Gel filtration is two words not one.17. Figure 1C, how were the flavins visualized?18. Line 144: Remove period after 'U/mg'.19. Line 165: BS3 is used but not explained until the methods section. For all non-standard abbreviations please define abbreviation at time of first use. This is particularly important for this abbreviation, which appears to be the abbreviation BS with a reference to footnote number 3 (non-existent).20. Line 182, may I suggest 'dehydrogenase coupled' or 'dehydrogenase connected' as conducting does not quite fit here.21. Figure 3C. This figure is very attractive, but the lettering does not quite match the ribbons with respect to color. Please use the dropper tool to achieve a match. This request is made only because the figure is otherwise so attractively colored. I have not devoted the same attention to average figures. Also please say what software was used to generate the figures, especially if the software is provided for free (support the authors with generous credit).22. Numbering of the supplemental figures and tables is very confusing. Please assemble these in a single document and use each numerical designation only once. E.g.: please only call one figure S1A. Please use the term 'figure' for all graphics and reserve the term 'table' for all numerical and otherwise tabulated information .Line 281 advertises the existence of another figure, but no figure number is given, my guess is that Figure S5 is intended.23. Line 303, please provide a literature reference for the 'single nucleotide exchange' method. Some of the amino acid substitutions described would appear to require that more than one nucleotide be changed, so I infer a clever new method.24. Line 304 'supplementary File' please refer to a specific supplementary table or figure.25. Line 320, the Δ205 notation is used, but I believe R205A is intended. Please reserve the Δ symbol for deletions. Similarly, on line 332, are these deletions? Substitutions are described in the supporting table.26. Line 331, 'farer' should possibly be 'farther'?27. Lines 340-341, Thank you for this. Please cite other examples in the literature.28. Line 468: 'red and orange' should be 'orange and red' to agree with the oxidation states listed earlier in the line.29. Lines 667, 671. Duplicates of the same reference.30. Line 743, please provide complete reference information31. Lines 776 and 782. Duplicates of the same reference.32. Lines 621, 622, 643, 795, flawed carriage return.33. Page 34: why is the first figure in the supplement Figure 3? Please call it figure S1 . Why is a table also called Figure 3?34. Page 42: Table A (primers). What distinguishes the lower-case nucleotides from those listed in upper-case? I could not see that the reverse primers were the reverse complements of the forward primers. Please explain.35. Page 5, why does purification need to be done anaerobically? Was the rest of structure determination handled anaerobically as well?36. In Figure 1B, why does the native PAGE of (Ldh-EtfAB)2 show the bands around 220 and 280 kDa instead of 253 kDa (Line 121). What are these bands? The authors should improve the quality of bands shown in the native PAGE by performing the analysis again.37. As the complex can easily dissociate, has the Kd value been estimated?38. Why did the authors not try to determine individual structures of EtfAB? By adding NAD+, would it be possible to obtain the X-ray determined structure in addition to ALphaFold calculations?39. Line 147, typo "its highest.."40. Line 148, I am curious about the data showing that the FBEC activity was inactive at pH 5. In real situations, what is the cellular pH of A. woodii? I think such microbes would have pH around that range. If FBEC is inactive at pH5, how can it conduct its reactions inside the cell?41. Figure 2, explanation regarding pH-dependent activity profiles should also be given.42. Were the assays done under the optimized conditions giving the highest activities?43. Page 8, how can one be sure that the cross-linking by BS does not create artifacts?44. Page 13-14, a diagram explaining assays in Table 2 should be helpful for the reader to understand.45. Page 14, "..variant consisting of the D122A, D124A, T125G, Q127G, V128A and 311 P130A substitutions shows a lower overall FAD amount of 1.7……….oxidoreductase activities were reduced to 7% and not anymore measurable, respectively. Likewise, the NADH:DCPIP oxidoreductase activity, measuring the hydride transfer from NADH to b-FAD, was also abolished. Both findings can be explained by the 316 expected strong decrease of the b-FAD content……"Was the decrease in activities mentioned proportional to the amount of FAD lost? One can calculate whether activities detected are according to the % FAD retained?46. Page 14-15, for the abolishment of the hydride transfer activity in the NADH:DCIP assays, have the authors tried to measure direct hydride transfer to b-FAD. Can this be done by monitoring FAD reduction under anaerobic conditions?47. Line 367, the iron in [ΔFe/S-domain] and [ΔFe/S] variants are completely lost, but why does the overall yield increase 4-fold.48. Line 442, regarding the statement that external Fdred binding may induce conformational changes to reduce their distance to ca. 14 Å, any evidence to support this?49. Line 464, regarding the statement that Glu37 and Glu139 are only found in the Ldh-EtfAB complex and these residues may destabilize the electron-rich reduced state of flavin, it would be better to have a reference to endorse this statement.eLife. Your revised article has been evaluated by Philip Cole (Senior Editor) , a Reviewing Editor, and an external reviewer.Thank you for resubmitting your work entitled "Structure-based electron-confurcation mechanism of the Ldh-EtfAB complex" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:Reviewer #1 (Recommendations for the authors):The manuscript has been improved and read better than the previous version.Perhaps, the authors may consider the following suggestions to make them clearer.1. I find Figure 1 confusing and too crowded. The authors may consider drawing Two separate panels (left and right) to explain different scenarios of confurcation and bifurcation. Do not mix them in the same figure.2. The authors may consider adding more responses to reviewers as a brief explanation of the manuscript as well. This will be clearer to readers.Reviewer #3 (Recommendations for the authors):The authors have carefully revised the manuscript according to the reviewers' suggestions and advice. The reported results are of interest and add to our understanding of electron bifurcation.The authors may notice that the sentences on lines 174-177, 182-185, and 269 should be better re-written for the sake of clarity. General clarifications:The manuscript needs considerable improvement with an eye to the readers who are not familiar with the electron bifurcation and other known electron bifurcating complexes.The term shuttle is used to mean one domain of the ETF. Please provide a definition at the time of first use, so that this is clear. Specific comments:1. It is interesting to see the impact that alphafold can have on experimental structure analysis. However, as a general comment, the authors tend to use the alphafold structures almost uncritically, as if they were experimentally determined. The manuscript should refer to the quality indicators given by alphafold and indicate how reliable the models are in particular around the regions that are mechanistically discussed in the text.We have used as input only the sequence of EtfA and did not work with any template. As result we took the pdb file with the highest quality values . The quality plot suggests a reliable prediction throughout the chain (except for the last 30 residues), that also includes the segment forming the interface. The rms deviation between the Alphafold EtfA model and the experimental model was 1.31 Å for the EtfA base (domain I) and 0.98 Å for EtfA shuttle domain (domain II) indicating a high agreement. A different angle between the EtfAB base and the EtfAB shuttle domain was expected in comparison to that of the experimentally analyzed D-state as EtfB and Ldh was not included in the AlphaFold calculation. The new interface between the EtfAB base and the EtfAB shuttle domain is plausible. Several hydrophobic and hydrophilic interactions are formed shown in the new Figure 5 – Supplement 1C. The buried area is ca. 700 Å2.2. The last paragraph of the main text (lines 446-460) should be mostly re-written and clarified. The sentence "The midpoint redox potential of the pyruvate/lactate pair is -190 mV, which is substantially lower than that of the crotonyl-CoA/butyryl-CoA pair of 10 mV, which turns a bifurcation into a confurcation event" requires a better explanation as, here, we are at the heart of the functional and structural principles governing the LDH complex. Figure 7B remains thereby difficult to be fully understood. Likewise, the sentence "When assuming that the redox potentials of the electron carriers between b-FAD and lactate also differ by ca. 180 mV the microenvironment of a-FAD and d-FAD should be distinguished between Bcd-EtfAB and Ldh-EtfAB ." remains honestly quite obscure to me. Likewise, the text should improve the discussion about the role of Gly37 and Gly139 and why the flavin of LDH should feature a lower redox potential compared to the analogous flavin of Bcd.We have completely rewritten the final paragraph of the manuscript.3. The manuscript should explain more clearly and from the beginning, the difference between confurcation and bifurcation. Likewise, the introduction should be accompanied by a scheme depicting the overall mechanism of electron bifurcation/confurcation in general and with reference to the reaction by the LDH system.We have added a new figure 1 into the introduction part involving also the Ldh-EtfAB complex as suggested.4. The abstract should be improved. Specifically, the text at lines 38-42 will be difficult to follow, at least for the casual reader.We modified lines 38-42 in the abstract but there is not much space for explaining.5. Figure 2 legend, explain the meaning of CODH.Carbon monoxide dehydrogenase (CODH) was used to keep the amount of reduced ferredoxin in the FBEC assay constant. The term CODH is now explained in the figure legend. The importance of CODH in the assay is explained in the material and methods part. 6. Line 189, the meaning of "promiscuous" EtfAB should be described explicitly.“Promiscuous” means in this context that several dehydrogenase types can associate with the same EtfAB complex. In the manuscript we have replaced the word “promiscuous”.7. Line 306, please describe where the residues 122-130 are located in the structure of the complex and why they define an electron-bifurcating system. Why are these residues making the difference between bifurcating and non-bifurcating enzymes?In non-bifurcating EtfAB the b-FAD is replaced by an AMP. Residues 122-130 flank the isoalloxazine ring in bifurcating EtfAB, which is absent in non-bifurcating EtfAB. We showed the position of residues 122-130 in Figure 7. The residues relevant for FBEB are described in the review of Buckel and Thauer, 2018. This review is cited at this position.8. The supplement figures of Table 1 should probably be moved to the main text as they are crucial for understanding the rationale of the mutagenesis experiments.We moved this figure into the main text after Table 1 as figure 7.9. Line 322, for the analysis of the catalytic role of Arg205, the authors may refer to recently published work (doi.org/10.1016/j.jbc.2022.101733).We integrated this reference into the text.10. Line 180 and throughout, CarCDE and Fix-EtfABX should be described in more detail as the authors often refer to them for their structural and functional comparisons.We made a supplementary figure showing the structures of Bcd-EtfAB, CarCDE and Fix-EtfABC and Ldh-EtfAB . One EtfAB unit is in the same orientation in all structures.11. Line 437, can the authors comment on the proton sources that couple protonation and reduction of the flavins.There is no obvious adjacent amino acid suitable for accepting/donating a proton from/to N5 of b-FAD. There is, of course, Arg205, which is not a suitable proton donor but it is in direct contact to N5 and the bulk solvent and may act as mediator. It is unclear yet whether NADH binding completely shields N5. There might be an access from the NAD binding site.12. Figure 7, the legend states that the two-electron reduced FAD is shown in orange but the drawing shows it colored in red.Thank you, we have corrected.13. Figure 7 legend, what is an "escapement" mechanism? Explain.We explained the escapement mechanism in the legend of Figure 1B.14. The figure supplement 1 of Figure 4 does not show the residues listed in the figure legend .In this figure the overall view of the interface is more important than individual residues. We changed the figure in a way to take into accounts the comment and to keep the figure clear.15. Line 102, rephrase, for example replacing 'holded' by 'held'. Corrected.16. Line 125: Gel filtration is two words not one.Corrected.17. Figure 1C, how were the flavins visualized?Flavins were visualized under UV light. We added a sentence in the figure legends.18. Line 144: Remove period after 'U/mg'.Corrected.19. Line 165: BS3 is used but not explained until the methods section. For all non-standard abbreviations please define abbreviation at time of first use. This is particularly important for this abbreviation, which appears to be the abbreviation BS with a reference to footnote number 3 (non-existent).A. woodii was determined from a sample cross-linked with bis (sulfosuccinimidyl) suberate (BS3) which is one of the most used agents for this purpose. The upper 3 belongs to the name of the cross-linker.The cryo-EM structure of the fragile Ldh-EtfAB complex from 20. Line 182, may I suggest 'dehydrogenase coupled' or 'dehydrogenase connected' as conducting does not quite fit here.We changed to ‘dehydrogenation-connected’. We wish to say that the shuttle a-FAD is in productive electron transfer distance to the d-FAD of the dehydrogenase.21. Figure 3C. This figure is very attractive, but the lettering does not quite match the ribbons with respect to color. Please use the dropper tool to achieve a match. This request is made only because the figure is otherwise so attractively colored. I have not devoted the same attention to average figures. Also please say what software was used to generate the figures, especially if the software is provided for free (support the authors with generous credit).We improved this figure, adapted the letter and the colors. We mentioned in line 616 that Chimera was used to produce this figure.22. Numbering of the supplemental figures and tables is very confusing. Please assemble these in a single document and use each numerical designation only once. E.g.: please only call one figure S1A. Please use the term 'figure' for all graphics and reserve the term 'table' for all numerical and otherwise tabulated information .Line 281 advertises the existence of another figure, but no figure number is given, my guess is that Figure S5 is intended.eLife rules.We collected all Figure supplements and one supplementary figure in one file and tried to follow the 23. Line 303, please provide a literature reference for the 'single nucleotide exchange' method. Some of the amino acid substitutions described would appear to require that more than one nucleotide be changed, so I infer a clever new method.This kind of amino acid substitutions were previously described in Demmer et al. 2018 (doi: 10.1002/1873-3468.12971). We added the reference.24. Line 304 'supplementary File' please refer to a specific supplementary table or figure.eLife manuscripts a related handling of the Supplementary file. If this is not possible we will find a solution.Supplementary file 1 stands alone and does not really fit to a specific figure or table. We saw in other 25. Line 320, the Δ205 notation is used, but I believe R205A is intended. Please reserve the Δ symbol for deletions. Similarly, on line 332, are these deletions? Substitutions are described in the supporting table.We changed ∆205 and ∆189 to R205A and D189A.26. Line 331, 'farer' should possibly be 'farther'?Corrected.27. Lines 340-341, Thank you for this. Please cite other examples in the literature.We cited the structural papers of the EtfB family.28. Line 468: 'red and orange' should be 'orange and red' to agree with the oxidation states listed earlier in the line.Corrected.29. Lines 667, 671. Duplicates of the same reference.These are two different references from the same authors.Corrected.30. Line 743, please provide complete reference informationMüller, V. (2008) Bacterial Fermentation. Encyclopedia of Life Sciences. Chichester:John Wiley and Sons, United Kingdom31. Lines 776 and 782. Duplicates of the same reference.Corrected.32. Lines 621, 622, 643, 795, flawed carriage return.Corrected.33. Page 34: why is the first figure in the supplement Figure 3? Please call it figure S1 . Why is a table also called Figure 3?Because the table belongs to Figure 3. We renamed several figure supplements.34. Page 42: Table A (primers). What distinguishes the lower-case nucleotides from those listed in upper-case? I could not see that the reverse primers were the reverse complements of the forward primers. Please explain.The applied site-directed mutagenesis method does not require a reverse complement for ligation because it’s a blunt-end ligation. Therefore, the primers have also no reverse complement regions. Back-to-back primer design methods have the advantage of transforming non-nicked plasmids. In addition, because the primers do not overlap each other, deletions sizes are only limited by the plasmid and insertions are only limited by the constraints of modern primer synthesis. In the first step we amplified our genes using primers described in Table 1 – Supplement 1. The second step involves incubation with an enzyme mix containing a kinase, a ligase and DpnI. Together, these enzymes allow for rapid blunt-end circularization of the PCR product and removal of the template DNA. The last step was the transformation into chemically competent cells.35. Page 5, why does purification need to be done anaerobically? Was the rest of structure determination handled anaerobically as well?2 free buffers. Kinetic FBEB or FBEC experiments have also to be done under anaerobic conditions as O2 competes with ferredoxin.Expression and purification have to be done under anaerobic conditions because the [4Fe-4S] cluster is highly oxygen-sensitive. The grid freezing procedure, finished in ca. 1 min, was performed on air by using O36. In Figure 1B, why does the native PAGE of (Ldh-EtfAB)2 show the bands around 220 and 280 kDa instead of 253 kDa (Line 121). What are these bands? The authors should improve the quality of bands shown in the native PAGE by performing the analysis again.First, we have to apologize using a picture of such a bad quality gel. We repeat the native PAGE experiments in the revised manuscript. The clear bands of the new gel allowed us to recalculate the sizes of the Ldh/EtfAB complex in the native page, which are approximately ≈150 kDa and ≈265 kDa. It corresponds to the sizes of a monomeric and dimeric form. Due to its fragility the Ldh-EtfAB complex is partially dissociated in the native PAGE. In addition, gel filtration profiles clearly indicate the partial dissociation of the Ldh-EtfAB complex into Ldh and EtfAB. The migration of the Ldh-EtfAB complex as two different fragments with different sizes was already shown in the first publication of the purified complex  and is obviously independent of the expression and purification procedure. We added Figure 1 – supplement 1 to show the disassembly.37. As the complex can easily dissociate, has the Kd value been estimated?A. woodii enzyme and does not bring us forward in understanding FBEB/FBEC.No, this number would be only relevant for the 38. Why did the authors not try to determine individual structures of EtfAB? By adding NAD+, would it be possible to obtain the X-ray determined structure in addition to ALphaFold calculations?Acidaminococcus fermentans EtfAB not only in the substrate-free form but also by soaking NAD+/NADH. But we always obtained the same non-physiological conformation. Moreover, we worked several years on the Bcd-EtfAB complex of Clostridium difficile to characterize the B state by X-ray and EM structural analysis but without success. Even when we had determined an EtfAB structure you could still be sceptic as the two flexible linkers might still not adjust the correct orientation between the EtfAB base and the EtfAB shuttle domain due to crystal lattice effects. Because of the non-success we thought that the correct B-conformation is only short-living and when attainable at all, then in the complete (Ldh-EtfAB)2 complex. Therefore, we focused on the (Ldh-EtfAB)2 complex in this work. The plausible B-state obtained with AlphaFold2 later on was a big surprise for us.Structures of non-bifurcating and bifurcating EtfAB are already available in a physiologically incorrect B-like conformation. Previously, we did structure determination of 39. Line 147, typo "its highest.."Corrected.40. Line 148, I am curious about the data showing that the FBEC activity was inactive at pH 5. In real situations, what is the cellular pH of A. woodii? I think such microbes would have pH around that range. If FBEC is inactive at pH5, how can it conduct its reactions inside the cell?A. woodii has been not determined. However, the cellular pH of anaerobes is indeed not kept constant at around pH 7.0. Studies of cellular pH values in closely related species of A. woodii, were performed by Menzel and Gottschalk (1985). The extracellular pH of Acetobacterium wieringae decreased from 7.0 to 5.0 during growth on sugars; accordingly, the intracellular pH changed from 7.1 to 5.5 ).The cellular pH of A. woodii. during growth on lactate. The extracellular pH of A. woodii decreased from 7.2 to 6.5 during growth on 80 mM lactate. According to A. wieringae, the internal pH of A. woodii growing on lactate should be around 7.3 to 7.0, which is optimal for the Ldh-Etf complex.We determined the extracellular pH change of 41. Figure 2, explanation regarding pH-dependent activity profiles should also be given.a value of His423, the potential catalytic base for proton abstraction from lactate. Its pKa might be higher than 6.0 toward the uncharged form in the protein interior.The FBEC activity has a sharp maximum around pH 7.0. Due to the complexity of the system this result cannot directly be interpreted in structural terms. It is certainly compatible with the pK42. Were the assays done under the optimized conditions giving the highest activities?Yes, all activity assays were performed under optimized pH and temperature conditions.43. Page 8, how can one be sure that the cross-linking by BS does not create artifacts?3 is frequently used (https://doi.org/10.1016/j.cbpa.2020.07.008) and, so far, realistic results were obtained . The Ldh-EtfAB revealed a highly plausible architecture of the D-state and there is no doubt on its relevance. We would, however, not exclude that the cross-linker preferably stabilizes the shuttle domain in the resting D-state and therefore prevents populations of the B-state. An FBEC activity test resulted in 2.1 ± 0.1 U/mg corresponding to 20% turnover. Thus, the structure and function of the Ldh-EtfAB complex is not severely changed by cross-linking, in particular, when considering that the additional cross-linking and purification steps are stress situations for the fragile protein complex.We cannot exclude that the crosslinking experiment creates artefacts. BS44. Page 13-14, a diagram explaining assays in Table 2 should be helpful for the reader to understand.We have explicitly written in 45. Page 14, "..variant consisting of the D122A, D124A, T125G, Q127G, V128A and 311 P130A substitutions shows a lower overall FAD amount of 1.7……….oxidoreductase activities were reduced to 7% and not anymore measurable, respectively. Likewise, the NADH:DCPIP oxidoreductase activity, measuring the hydride transfer from NADH to b-FAD, was also abolished. Both findings can be explained by the 316 expected strong decrease of the b-FAD content……"Was the decrease in activities mentioned proportional to the amount of FAD lost? One can calculate whether activities detected are according to the % FAD retained?The methods applied for measuring the FAD content is not sufficiently sensitive to quantify small b-FAD amounts. It has to be considered that 3 FADs are involved and errors of ca. 10% are possible. The FBEC activity of 7% suggests a b-FAD content < 10% which is within the error of the FAD determination. No serious statement about the proportionality between activities and the amount of FAD is possible.46. Page 14-15, for the abolishment of the hydride transfer activity in the NADH:DCIP assays, have the authors tried to measure direct hydride transfer to b-FAD. Can this be done by monitoring FAD reduction under anaerobic conditions?A.fermentans . We expect similar results for EtfAB of the Ldh-EtfAB complex.In principle, it is possible to monitor b-FAD reduction with NADH alone. However, we have not done this experiment for Ldh-EtfAB. This type of experiment has been performed for EtfAB of 47. Line 367, the iron in [ΔFe/S-domain] and [ΔFe/S] variants are completely lost, but why does the overall yield increase 4-fold.We were also surprised to see an increase in the purification yield of [ΔFe/S-domain] and [ΔFe/S] variants. The same finding was made by the CarCDE complex . We can only speculate that the production of Ldh-EtfAB becomes simplified when the [FeS] cluster has not to be formed. Its extraordinary flexibility might further delay the maturation process.48. Line 442, regarding the statement that external Fdred binding may induce conformational changes to reduce their distance to ca. 14 Å, any evidence to support this?No, we have no experimental evidence that supports this proposal. It is difficult to structurally characterize a FBEB enzyme in complex with an external ferredoxin as its binding is normally rather weak. It would be lucky to find a suitable model system. However, we have executed an Alphafold2 calculation with EtfA and EtfB (previously only with EtfA) and the Fd domain moved to EtfB. The distance between the [4Fe-4S] cluster and b-FAD is thereby shortened to ca. 14 Å. This result is not surprising when considering the high mobility of the Fd domain. Moreover, a related phenomenon was already observed twice in FBEB enzymes . It appears to be useful for the FBEB/FBEC reaction that the electron transfer pathway between the external ferredoxin and the bifurcating flavin is only formed for the short period of the reaction. The interruption might also be a tool to ensure that only desired ferredoxins can properly dock to the surface and induce the conformational changes required for electron transfer.49. Line 464, regarding the statement that Glu37 and Glu139 are only found in the Ldh-EtfAB complex and these residues may destabilize the electron-rich reduced state of flavin, it would be better to have a reference to endorse this statement.For example, the following reference are relevant ; Mothersole et al. 2019, doi: 10.1002/pro.3661).There are mutagenesis data of charged residues adjacent to flavins but they will neither validate nor falsify our conclusion. These mutagenesis experiments are extremely delicate as small secondary changes at the isoalloxazine ring or its surrounding overcompensate the effect of pure mutation. A strong argument for the importance for Glu37 and Glu139 is their strict conservation in bifurcating Ldh but not in membrane-spanning Ldhs. Two acidic amino acids flanking two isoalloxazine group is a very unusual feature in biochemistry.The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:Reviewer #1 (Recommendations for the authors):The manuscript has been improved and read better than the previous version.Perhaps, the authors may consider the following suggestions to make them clearer.1. I find Figure 1 confusing and too crowded. The authors may consider drawing Two separate panels (left and right) to explain different scenarios of confurcation and bifurcation. Do not mix them in the same figure.Amended legend for Figure 1:2 between the strong electron acceptor (N) and the medium electron donor (MH) has a higher absolute value than the negative ∆E3 between Fdox and MH. In other words, ∆E1 has to be positive resulting in a negative Gibbs free energy . As example, the electrochemical treatment and the thermodynamic scheme was provided for the reaction of the bifurcating Bcd-EtfAB complex, in which the b-FAD reduced by NADH endergonically donates one electron via a-FAD to d-FAD and then exergonically one electron to Fdox. (B) The reverse FBEC reaction (1) calalyzing two oxidation reaction (2+3) with the same electron acceptor (M). Here, positive ∆E3 has a higher absolute value than the negative ∆E2. In the Ldh-EtfAB reaction low-potential Fdred donates an electron to b-FAD via one [4Fe-4S] cluster in an endergonic reaction that is driven by the exergonic ET from d-FAD, via a-FAD to b-FAD•−. The generated b-FADH‒ transfers a hydride to NAD+. For the FBEB/FBEC process the first uphill ET step to b-FAD is reversed except when instanteneously pulled out by the second downhill ET step termed as escapement-type mechanism . This tight coupling implicates catalytic inactivity in the absence of one of the three substrates and prevention of short circuit reactions e.g. from b-FAD•− to a-FAD•− and undesirable side reaction of the highly reactive b-FAD•−”.“Figure 1. Thermodynamic basis. (A) The FBEB reaction (1). It involves two reduction reactions (2 + 3) using the same electron donor (M) of medium redox potential and two electron acceptors (N and Fd) of high and low redox potential. Fd can be only replaced by flavodoxin . Biochemical reactions are normally characterized by a pair-wise exchange of electrons between substrates that are protonated in their reduced state. FBEB occur when the positive difference redox potential ∆E2. The authors may consider adding more responses to reviewers as a brief explanation of the manuscript as well. This will be clearer to readers.Reviewer #3 (Recommendations for the authors):The authors have carefully revised the manuscript according to the reviewers' suggestions and advice. The reported results are of interest and add to our understanding of electron bifurcation.The authors may notice that the sentences on lines 174-177, 182-185, and 269 should be better re-written for the sake of clarity.Line 174-179 (now 172-174)“In contrast, non-bifurcating oxidoreductase-EtfAB complexes form a transient D-like state, which is beneficial as the EtfAB shuttle domain assembles with different dehydrogenase partners .”Line 182-185 (now 178-180)Figure 5 – video 1) that deviates from the previously proposed one .”“The EtfAB shuttle domain swings ca. 75° from the D-state into a B-state position (Line 269 (now 264-266)Figure 7) and in the B-state 4.5 and 10 Å apart from the previously proposed and AlphaFold2-calculated a-FAD position, respectively .”“D189 of EtfA is 11 Å apart from b-FAD (


Transmission of Human papillomavirus (HPVs) is faithfully associated with carcinogenesis of oral cavity and oropharyngeal cancers. Therefore, clinical researchers may need to generate customized antibodies for the upcoming ELISA-based analysis to discover rare but valuable biomarkers. The aim of study was to develop and generate a biosensor-based immunoassay for early screening HPV-related oral cancer via saliva rinse fluid analysis. A peptide fragment of high-risk HPV subtype 16/18 protein, E6 protein (HP-1 protein sequence 48–66), was designed and synthesized, followed by the generation of polyclonal antibodies (anti-HP1 IgY) in our university-based laboratories. The titer and specificity of antibodies were determined by enzyme-linked immunosorbent assay (ELISA), and the Surface Plasmon Resonance (SPR) biosensor-based method was developed. Kinetic analyses by SPR confirmed that this designed peptide showed a high affinity with its generated polyclonal antibodies. Saliva fluid samples of thirty oral cancer patients and 13 healthy subjects were analyzed. SPR indicated that 26.8% of oral cancer patients had higher resonance unit (ΔRU) values than normal subjects. In conclusion, we developed a biosensor-based immunoassay to detect HPV E6 oncoprotein in the saliva rinse fluid for early screening and discrimination of HPV-related oral cancer patients. The development of new tools of biofluid technology for biomarker detection in saliva is challenging. However, saliva-based examination is potentially useful for oral cancer screening, monitoring treatment response and developing personalized medicines. Two-dimensional polyacrylamide gel electrophoresis 2-D PAGE) and Mass Spectrometry based assays may not be suitable for application in clinical settings because of analysis needed in laboratory-based facilities and it being time-consuming for data interpretation ,2. There-D PAGE aGrowing evidence supports the role of HPV in the cause and response of oral cancers squamous cell carcinoma (OCSCC) and oropharynx squamous cell carcinoma (OPSCC) . Latent Clinical researchers may need to generate customized antibodies for the upcoming ELISA-based analysis at a university-based laboratory to discover rare but valuable biomarkers. Here, we present a streamlined procedure to develop the biosensor approach of the antibody-based immunoassay using the SPR technique. First, we designed and synthesized an antigen, a novel peptide fragment  of HPV 16 E6 protein . ChickenOur study was approved by the Committee on Human Research of the Veterans General Hospital . All participants provided written informed consent for the collection of samples and analysis. Saliva samples were collected from 30 oral cancer patients and 13 healthy subjects at the Department of Stomatology, Taichung Veterans General Hospital. For healthy subjects, inclusion criteria were aged > 18 years and with no history of neoplasm. From 2015 to 2017, we enrolled 30 advanced T3–4 oral cancer patients who were never treated according to the 7th AJCC on Cancer TNM staging system. The collected saliva was added with an enzyme inhibitor . The collection steps were as follows: 20 to 30 mL of sterile distilled water were used to rinse the oral cavity gently and discarded. Another 5 mL distilled water was used for 1-min gargling and then spitted to collect all of the saliva, and samples were placed in ice immediately. After adding the protease inhibitor, the collected saliva sample was centrifuged at 3000 rpm at 4 °C. The supernatant of the sample was extracted and stored at −80 °C.We designed and synthesized a peptide fragment E6 48–66 in HPV 16 E6 protein (named as HP-1 peptide) and used that as an antigen for immunization in chickens. The egg yolk polyclonal antibodies were integrated into the immunoassay to develop a biosensor using the surface plasmon resonance. HP-1 was modified and extended in length at peptide region 52 to region 62. This region is well documented and is a target for CD4 T-cell epitope . Solid-pImmunization procedures followed the terms of the Institutional Animal Care and Use Committee (IACUC). The immune response in Lai Hen chickens was that the antigen was boosted by a complete adjuvant . The adjuvant prevents rapid disintegration of the antigen, prolonging the antigenic exposure to the immune system and enhancing the response of immune cells. The immunized injection contained an emulsified mixture of 300 μg formed by 150 μg of antigen and 150 μL of complete adjuvant (in 1:1 ratio). The second injection was given 14 days later, and consecutive immunizations were performed at 3-week intervals until reaching a stable sera titer. At each boost injection, the serum titer was checked, and the animal with the highest titer was selected, and her eggs were collected for antigen separation. The liquid phase extraction separated antibodies from the egg yolk, following the method described by Polson . First, Titers of egg yolk anti-HP-1 IgY antibodies were determined with the indirect enzyme-linked immunosorbent assay (ELISA). Briefly, into microtiter wells on the ELISA plate, 100 μL of each HP-1 peptide standard and 100 mL chicken anti-HP-1 IgY antibody were pipetted. Dispensed into each well was 100 μL of goat anti-chicken-HRP conjugate reagent. The mixture was allowed to incubate at room temperature under shaking (750 rpm) for 90 min. Next, the incubation mixture was removed by flicking plate contents into a waste container. Wells were rinsed and flicked five times with wash buffer, and the plate was struck sharply onto paper towels to remove residual water. Then, the TMB reagent (100 μL) was added to each well, gently mixed for 10 s, and allowed to incubate at room temperature in the dark with mechanical shaking (750 rpm) for 20 min. Next, hydrochloric acid  was added to each well and gently mixed for ~30 s to stop the enzymatic reaction (making sure that the blue color had changed to yellow completely). Within 15 min, the 450 nm absorbance was measured for each well using a VERSAmaxTM microplate reader .The Kinetic/Affinity analysis was performed using the Biacore 3000 to study the kinetics (binding and dissociation rate) and affinity (binding strength) of interactions of the antigen–antibody complexes. The following experiments adopted a multi-cycle mode, in which the regeneration step was performed after dissociation of each injection sample to ensure similar conditions of chips before each sample injection. The following experiment aimed to investigate the interactions between anti-HP-1 IgY antibody and E6 protein . Anti-HP-1 IgY antibodies diluted in serial concentrations were used as analytes. The highest concentration was 10.00 μg/mL, and from that diluted serially to 5.00 μg/mL, 3.33 mg/mL, 2.50 mg/mL, 2.00 mg/mL, and 1.66 μg/mL. Induction spectra were detected with the E6 protein immobilized on the biosensor chip.Initially, EDC/NHS was used to activate the surface of the sensor chip at a final concentration of 0.2 μg/mL, and anti-HP- 1 polyclonal antibody were covalently bound to the surface of the chip . The final EA masked the remaining activating sites. A sensorgram was generated by reacting a 1⁄2 diluted saliva sample from the 43 subjects as analytes. Anti-HP-1 antibodies (IgY) were immobilized on the sensor chip, and the saliva sample was tested in the solution that flew through the channel on the chip for monitoring the to-be-detected interactions. The sample mixture was diluted with an HBS buffer and analyzed with Biacore 3000 at a flow rate of 20 μL/min.The peptide sequence of HP-1 was HN2-EVYDFAFRDLCIVYRDGNP-COONH2, and the theoretical molecular weight was 2292.08 Da. RP-HPLC purified the crude peptide mixture, and the retention time (Rt) of the HP-1 peptide was determined as 14.73 min. The solution collected at 14.73 min was freeze-dried, and its molecular mass was determined as 2293.5 m/z by MALDI-TOF MS. These results indicated the successful synthesis and purification of HP-1 peptide a,b.The highest concentration of egg yolk antibody was 250 μg/mL, which was diluted serially to 125 μg/mL, 62.5 μg/mL, 31.25 μg/mL, 15.625 μg/mL, 7.8125 μg/mL, and 3.90625 μg/mL .The detected responses of egg yolk antibodies ranged from 0.25 mg/mL to 0.0625 μg/mL were found to be dose-dependent. In addition, we found an excellent linear relationship between the antibody and antigen was within the concentration of 1.953125 μg/mL. Therefore, in the following ELISA analysis, the antibody concentration was set up at 125 μg/mL.−1 s−1 and 1.191 × 10−4 s−1, respectively  were tested using half diluted saliva as analytes, in which anti-HP-1 IgY antibodies had shown immobilization at the sensor chip. The sensorgram revealed that some patients’ saliva had interacted more strongly with antibodies. This finding indicated that these few patients had E6 protein present in their saliva samples. When the value of RU was set at 18.694, we found both positive and negative results . With the upper limit as the cutoff level, it was found that all normal subjects with values < 18.694 RU were classified as negative results, and 26.7% of oral cancer patients (stage III + IV) showed positive results .In this study, we designed and synthesized a specific candidate peptide, HP-1, for HPV E6 protein as a salivary biomarker for oral cancer. The kinetic analysis demonstrated a high affinity between our peptide immunized IgY antibody and the E6 protein. Thus, we anticipate that this self-generated IgY can provide a fundamental immunoassay model to develop tools for detecting salivary biomarkers at university-based laboratories, particularly for the rare but valuable peptides as candidate biomarkers.In addition, we successfully established a biosensor/biochip immunoassay to detect the presence of HPV E6 protein in the saliva of oral cancer patients. Empirically, we analyzed 30–40 samples for testing our biomarkers and biosensor in this study. Although the small sample size can lead to a wide range of confidence intervals and inaccuracies in statistical hypotheses testing, it showed that 26.8% of advanced oral cancer patients had higher levels of E6 protein in their salivary samples when considered at a cutoff level of 18.694 ΔRU. Following this result, one-fourth of the HPV E6 positive rate is compatible with previous reports of incidence of HPV-related oral and oropharyngeal cancer patients ,18. The The scheme of detecting salivary biomarkers for oral cancer is a promising step forward for individualized medicine. Bio-fluids, such as saliva, could contain tumor-related DNAs and proteins related to the diseases ,20. SeveHowever, targeting HPV oncoproteins, as an oral cancer biomarker facilitates new approaches in salivary-based assays for early diagnosis, clinical staging, and prediction of treatment outcomes . In the Compared to HPV DNA, E6/E7 protein expression might be more directly associated with human cancer risk. The reason might be that the presence of HPV DNA is not enough to prove the causality of malignant transformation. Mainly HPV E6/E7 oncogene expression is considered necessary for carcinogenesis. Therefore, the lack of HPV E6/E7 oncogene expression represents the carcinogenic causality of HPV does not exist . Based oCurrently, there are several tests available for diagnosing HPV-related OPSCC. HPV DNA and mRNA assess HPV-related infection or tumors by PCR and Rt-PCR in the paraffin-fixed tissue specimens. The main strength of those examinations is high sensitivity and specificity; however, they are unpractical for usage in clinical applications . The altApplication of IgY is of potential use for treatment and cancer-related research and in developing immunotherapy against surface tumor markers in cancer cells. Several studies reported on the capacity of IgY-based immunoassay for diagnostic purposes and research ,32. ThisSurface plasmon resonance (SPR) techniques have a high potential for clinical applications to obtain a more efficient salivary-based analysis. The primary measuring mechanism of SPR immunoassay is detecting changes in the optically refractive index of the metal chip based on interactions between antigen and antibody that immobilize products in samples. SPR showed some advantages in proteomic analysis . This SPWe reported a biosensor/biochip-based immunoassay for identifying saliva biomarkers in oral cancer patients. It is potentially helpful for oral cancer screening, monitoring treatment response, and developing personalized medicines. Furthermore, such an approach of IgY-based internal detection is also suitable for biomarker detection of new pandemic viruses (such as COVID-19), as the method is cost-effective and is fast in implementation and analysis. The primary goal is to develop a biosensor-based assay to detect salivary biomarkers of oral cancer patients, a rapid, efficient, and non-invasive diagnostic tool.This manuscript builds on our study to identify salivary biomarkers based on immunoassay biosensor/biochip techniques. The U.S. patent (US 10379123) of this technique has been filed.


The nanofibrillar morphology, due to the well-defined individual nanofibers separated by microgaps and micropores and a better organization of the carbon network, leads to a larger specific surface area and higher conductivity, which can allow more efficient substrate transport and better bacterial colonization with greater relative abundances of Geobacter and Thermoanaerobacter, justifying the improvement of MFC performance.Anode modification with carbon nanomaterials is an important strategy for the improvement of microbial fuel cell (MFC) performance. The presence of nitrogen in the carbon network, introduced as active nitrogen functional groups, is considered beneficial for anode modification. In this aim, nitrogen-containing carbon nanostructures (NCNs) with different morphologies were obtained via carbonization of polyaniline and were further investigated as anode modifiers in MFCs. The present study investigates the influence of NCN morphology on the changes in the anodic microbial community and MFC performance. Results show that the nanofibrillar morphology of NCNs is beneficial for the improvement of MFC performance, with a maximum power density of 40.4 mW/m To taterials , N-dopedaterials , nitrogeaterials , self-niaterials , and nitaterials , were usaterials ,26,27,28aterials ,30,31. Tuel cell , a novelFurthermore, the use of mixed microbial communities in MFCs  gained a lot of attention due to their stability and high power production compared with pure cultures ,33. UndeIn our study, we synthesized polyaniline with different morphologies, which were then used as precursors for nitrogen-containing carbon nanostructures (NCNs). The resulting NCNs with different morphology were used as anode modifiers in an MFC system loaded with municipal wastewater, and the microbial diversity of the resulting biofilms formed on the anode surface was analyzed, along with that of the initial wastewater, through high-throughput short read sequencing. The present study investigates how the changes in NCN morphology and anodic microbial community affect MFC performance, which, to our knowledge, has not been reported in the existing literature.Two different synthesis methods used in our work allowed for the synthesis of polyaniline with different morphologies, one with near-granular-shaped nanoparticles (PANI-R) and another with nanofibrillar morphology (PANI-T). Direct carbonization of polymeric precursors (PANI-R and PANI-T) allowed us to obtain NCNs with different morphologies (PANI-R-900 and PANI-T-900). The SEM micrographs depicted in From energy dispersive X-ray analysis (EDX), the elemental compositions of all samples are summarized in Thus, in our work, we synthesized polyaniline with different morphologies, which was further used as a polymeric precursor for NCNs. The results prove that the direct carbonization of polyaniline nanostructures is an easy and economical process to obtain nitrogen-rich carbon materials, preserving the general features of the precursor’s morphology, with a smaller decrease in nanoparticle size, as can be seen in the SEM images and EDX analysis of the polymer precursors and their corresponding NCNs.−1 and ~1493 cm−1 are assigned to the C=C stretching vibration of the quinoid and benzenoid rings, respectively. The band at ~1306 cm−1 can be attributed to the C–N–C stretching vibration or to the π-electron delocalization induced in the polymer through protonation, while the band at 1248 cm−1 is attributed to the C–N+● stretching mode of the polaron structure, which is characteristic of the conducting protonated form of PANI [−1 can be assigned to a C-H bending vibration or the vibrations of the charged polymer units Q=NH+–B or B–NH+●–B (Q denotes the quinoid ring and B denotes the benzenoid ring), formed during protonation [In  of PANI ,35,36. Ttonation ,35,37.−1 and 700 cm−1 correspond to hydrogen sulfate counterions, which are characteristic of the conducting form of PANI [−1–800 cm−1 are attributed to the aromatic C–H vibrations. In addition to the above peaks, the spectrum of the PANI-R exhibits bands at 3238 cm−1 that are attributed to N-H stretching mode, and the absorption between ~3063 cm−1–2830 cm−1 corresponds to an aromatic C–H stretching vibration [−1, 1042 cm−1, 750 cm−1 and 688 cm−1, which can be associated with the different morphology of the samples, as reported previously [−1 corresponds to the skeletal C=C stretching vibration of the aromatic ring; 1042 cm−1 is due to the sulphonate groups attached to the aromatic rings [−1 and 688 cm−1 are related to the monosubstituted aromatic rings as terminal units or indicate more pronounced branching chains in the nanofiber samples [The bands at about 1080 cm of PANI . The banibration ,37,38. Ieviously . Thus, tic rings . The ban samples ,39,40.−1, except for the two broad bands with maxima at 1580 cm−1 and 1132 cm−1. The first one is assigned to the aromatic ring vibration mixed with C=N stretching vibration, and the second ones correspond to C–C and C–N vibrations, respectively. These bands are characteristic of carbon-like materials that have Raman G- and D-bands, which are not IR active. The presence of nitrogen in our carbonized samples, also confirmed by the EDX analysis, breaks the symmetry of the carbon network, and these bands become active in the FTIR spectra [The FTIR spectra of both carbonized samples show almost no peaks in the whole range of 400–4000 cm spectra ,42,43.2 atmosphere at a temperature of 900 °C . The carbonization of PANI-R and PANI-T is highlighted by the presence of two broad peaks corresponding to diffraction planes (002) and (101) of a disordered graphite phase [−1 ± 2.4 × 10−3, in comparison with 9.6 × 10−2 ± 2.1 × 10−3 for PANI-T-900. These sensitive differences in microstructural parameters between the two samples also show that the nanofiber morphology of PANI-T, which has a better periodicity of polymer chains, favored the formation of a more ordered graphite phase than the sample PANI-R.The X-ray diffraction patterns from te phase . To obtate phase . For bot2 is obtained in the case of anode modification with NCNs with nanofibrillar morphology, PANI-T-900, compared with 32.3 mW/m2 obtained for anode modification with PANI-R-900 and 18.8 mW/m2 for MFC with carbon cloth (CC) anode. The average values (four replicas for each modification) of open circuit potential (OCP), power and current density and internal resistance for MFC, which used the same modifier for the MFC anode, PANI-R-900 and PANI-T-900, are summarized in In our study, 1 kΩ external resistance was used for anode biofilm inoculation. For the polarization measurements, the optimal resistance, defined as the external resistance for producing maximum power in MFCs, or a high external resistance (1 kΩ), which can facilitate a more compact structure of the biofilm, can be used ,51. As rThe polarization measurements show that the internal resistance of the cell followed the order: CC (1272 Ω) > PANI-R-900-modified CC anode (839 Ω) > PANI-T-900-modified CC anode (782 Ω) see . Accordi3V4 region of the bacterial 16S rRNA gene from the total biofilm DNA samples was amplified and sequenced using NGS  technologies.The microbial community formed on the surface of the PANI-R-900- and PANI-T-900-modified CC anodes, as well as the microbial community of the initial wastewater, were characterized through metabarcoding techniques. The Vp < 0.05) in the anode biofilms from the initial wastewater. Pielou’s evenness values did not differ greatly, although a slight decrease was observed in the case of the PANI-T-900-modified anodes, indicating a larger variation within more closely related taxa when compared to the PANI-R-900-modified anodes.Results show that the operating conditions as well as the anode type determined an enrichment of select taxa from the initial wastewater. Regarding alpha-diversity metrics , there wArcobacter sp. (22.9%), Pseudoarcobacter sp. (14.81%), and unclassified Arcobacteraceae (17.83%), the family being one of the most abundant in raw sewage and wastewater samples [Gammaproteobacteria (19.19%) and Bacteroidia (16.22%), also commonly present in wastewater [Feature taxonomy at the class level underwent major changes when comparing the most abundant taxa from the initial wastewater to the anode biofilms . The ini samples . Other astewater . Campylobacteria and Gammaproteobacteria relative abundance decreased greatly, while Bacteroidia abundance increased, more so in the case of PANI-T-modified anodes, 28.22% compared to 23.9% for PANI-R-900. The most enriched classes for both anode types were Thermoanaerobacteria , Desulfuromonadia , Clostridia , Synergistia, Desulfobacteria and Desulfovibrionia. In the anode biofilms, Thermoanaerobacteria class are more commonly classified into Clostridia, the only genus present being Thermoanaerobacter  and Methanomassilicoccales , a characteristic of anaerobic systems, could bring added value to the MFC system through methanogenesis from acetate [MFC operation determined an enrichment of  acetate .Geobacter genus, the type genus studied for its exoelectrogenic capabilities [Thermoanaerobacter sp. (as mentioned previously) and Macellibacteroides [At the genus and family levels , resultsbilities ,62, had teroides  were morTrichloromonas [Rhodocyclaceae and Comamonadaceae families [Other known EET-capable bacterial taxa enriched in the case of both anode modifiers used are oromonas  and membfamilies .Direct carbonization of polyaniline (PANI) as an N-containing aromatic polymer with N covalently bonded in polymeric precursors is an efficient way to prepare N-containing carbon nanostructures, most frequently by preserving the morphology of the PANI precursor.2SO4 under stirring conditions at room temperature for 30 min. A total of 0.1 M ammonium persulfate was dissolved in 50 mL of 0.1 M H2SO4 and pre-cooled before adding to the monomer solution drop by drop. The polymerization solution was allowed to react overnight at room temperature. The resulting precipitate was collected and washed several times with deionized water and methanol. Finally, the product was dried at 60 °C overnight and indexed as PANI-R. The second procedure for the synthesis of polyaniline nanostructures was carried out using a template-free self-assembly method in an appropriate mixed solution of 0.4 M ethanol and 0.4 M acetic acid, as reported in reference [Two different synthesis methods were used to obtain different morphologies for PANI precursors. In the first method, polyaniline nanostructures were prepared by the chemical oxidative polymerization of an aniline monomer in acid media using ammonium persulfate as the oxidizing agent, following a modified procedure of an adapted method previously reported by Rezvani et al. . In a tyeference . SeparatNCNs were obtained after carbonization of PANI-R and PANI-T at 900 °C under nitrogen flow, with a 5 °C/min heating rate and 1 h holding time at 900 °C, followed by free cooling to room temperature under nitrogen flow. The NCN samples were indexed as PANI-R-900 and PANI-T-900, respectively.The morphological characteristics and elemental composition of PANI nanostructures and corresponding NCNs were analyzed using scanning electron microscopy , using a secondary electron detector and 5 kV accelerating voltage), coupled with an energy dispersive X-ray spectrometer . Quantification of the EDX spectra was performed using the Kα lines by means of the Bruker ESPRIT Quant v2.3 software .−1, with a resolution of 4 cm−1 on a Varian 3100 Excalibur spectrophotometer . The samples were analyzed by attenuated total reflection (ATR) using a Pike MIRacle  accessory. The crystalline features of PANI nanostructures and corresponding NCNs were characterized by X-ray diffraction (XRD) using a Bruker D8 Discover diffractometer (CuKα = 1.54 Å) in symmetric geometry. The data were collected in the interval of 2θ = 10–80° with an angular step of 0.02° at ambient conditions.Fourier transform infrared (FTIR) spectroscopy analysis of the PANI nanostructure samples was performed by recording 128 scans in the range of 600–4000 cmv/v) containing 0.5% Nafion was prepared. After homogenization in an ultrasonic bath for 30 min, the ink was sprayed onto the carbon cloth surface using an airbrush. The loading of NCNs on carbon cloth for all MFCs was ~1.1 mg/cm2. For Pt-containing cathodes, commercial Pt catalyst  ink mixed with a chemical binder (5% Nafion solution) was sprayed over carbon cloth. The anolyte consisted of municipal wastewater  supplemented with 1 g/L of acetate, and phosphate buffer  was used as the catholyte. The anode chamber was hermetically sealed, while the cathode chamber was purged with bubbling air. Cell voltages across the external resistor were recorded using a Pico Data Logger ADC-24  and a personal computer. For each modification, four identical MFCs with PANI-R-900- and PANI-T-900-modified CC anodes were operated simultaneously and compared with MFC using a CC anode as a controller. The polarization curves and power output were obtained by varying the external resistor from 1 MΩ to 40 Ω. The open cell potential (OCP) was measured after 120 min. At each resistance, MFC ran for at least 10 min to ensure that a stable power output had been achieved. Current (I) was calculated as I = V (cell voltage)/R , and power (P) was calculated as P = I × V. Both current density and power density were calculated based on the surface area of the anode.The double chamber is commonly used in laboratory research for the investigation of new substrates, electrode materials, membranes or types of microbial communities, and it is known for its low power generation because of its complex design and high internal resistance ,68. A dohttps://en.novogene.com/, accessed on 15 march 2022). Samples were amplified using universal primers for the V3-V4 region of the bacterial 16S rRNA gene (sequencing depth—100 k tags). The PCR products were selected by 2% agarose gel electrophoresis . Equal amounts of PCR product from each sample were pooled, end-repaired, A-tailed, and further ligated with Illumina adapters. Libraries were sequenced on a paired-end Illumina platform to generate 250 bp paired-end raw reads. Data analysis was performed in-house using the QIIME2 toolkit [Anode biofilm and initial wastewater total DNA were isolated using the Dneasy PowerSoil Kit  according to the manufacturer’s protocol, including the additional incubation step at 70 °C before vortexing samples. DNA, RNA, and protein quantification were done using a Qubit 4.0 and the dsDNA BR , RNA HS , and Protein  Assay Kits. Triplicate samples were selected for each anode type  and initial wastewater based on quality and DNA concentration. Sequencing was externalized towards Novogene ( toolkit . Sequenc toolkit  was used toolkit . A class toolkit , for the2 was obtained in the case of anode modification with PANI-T-900, which has a nanofibrillar morphology, being 1.25 times higher when compared to the anode modified with PANI-R-900 and 2.15 times higher than the MFC using the CC anode. The internal resistance reduction also resulted in the improvement of power generation. MFC operation determined the enrichment of various taxa, resulting in community profiles that differed greatly from the initial wastewater. The dominant taxa in the anode biofilms were part of genera and families associated with extracellular electron transfer, such as Geobacter, Thermoanaerobacter, Rhodocyclaceae, and Comamonadaceae. The use of PANI-T-900-modified CC anodes resulted in greater relative abundances for members of Geobacter and Thermoanaerobacter, justifying the MFCs’ better power generation. The majority of taxa identified were anaerobic, while bacteria from the Desulfobacterota phylum indicated the occurrence of sulphate reduction. The presence of methanogenic Archaea in the biofilms could indicate the synthesis of methane, a possible value-added byproduct, throughout the process. Since the experimental results proved that both modifications with NCNs lead to a better performance of MFCs, polyaniline-derived nitrogen-containing carbon nanostructures can be considered a very good material for MFC anode modification, especially due to the simple and economical process that can allow the introduction of nitrogen functionality on the anode surface. Further research would be necessary to undertake in order to correlate the change in nitrogen functionality with NCN morphology.In this paper, polyaniline with different morphologies was used as a precursor for the synthesis of nitrogen-containing carbon nanostructures. The NCNs with different morphology were considered for anode modification, and the resulting MFC performance was studied and compared with MFC with carbon cloth anode as controls. The maximum power density of 40.4 mW/m


Today, the development of nanomaterials with sensing properties attracts much scientific interest because of the demand for low-cost nontoxic colloidal nanoprobes with high sensitivity and selectivity for various biomedical and environment-related applications. Carbon dots (CDs) are promising candidates for these applications as they demonstrate unique optical properties with intense emissions, biocompatibility, and ease of fabrication. Herein, we developed synthesis protocols to obtain CDs based on o-phenylenediamine with a variety of optical responses depending on additional precursors and changes in the reaction media. The obtained CDs are N-doped  less than 10 nm spherical particles with emissions observed in the 300–600 nm spectral region depending on their chemical composition. These CDs may act simultaneously as absorptive/fluorescent sensing probes for solvent polarity with  In the past years impressive progress in sensing technologies has been achieved due to the development of spectroscopic and microelectronic devices along with success in synthetic methods including organic and/or inorganic materials and nano composites . This ha2+ that have been found to affect the activity of coenzymes which influence Alzheimer’s disease when present in cells [Considering biomedical applications and the food industry, sensing probes should also be biocompatible and eco-friendly, demonstrate a stable response under changing media and intense/continuous light exposure, and allow incorporation in various matrices for solid-state sensors. All of the above-mentioned criteria are met by carbon nanoparticles, making them a highly attractive subject of research. Due to their ability to support several optical centers, their excitation-dependent emission facilitates the sensitivity of chemical sensing via distinguishable spectral changes for different optical centers including ratiometric responses. The possibility to functionalize the surface of carbon nanoparticles allows a further increase in sensitivity to specific analytes such as nitrite and ascorbic acid  and enzyin cells . In addiin cells ,26. M. Zin cells . CDs synin cells . Furtherin cells  and for in cells . Howeverin cells .Herein, we developed synthesis protocols to obtain CDs based on o-PD that demonstrate a distinguishable response in absorption and photoluminescence to both solvent polarity and pH. It was achieved by a change of the synthesis parameters leading to a variety of chemical compositions and optical responses of nanoparticles. Based on these CDs, filter paper/cellulose test strips that can be used as a simple disposable sensing probe for colorimetric pH determination with a smartphone were fabricated.O-phenylenediamine , hydrochloric acid (38%), benzoic acid , and thiourea (>99%) were purchased from Sigma Aldrich. Rhodamine 6G was purchased from Sigma-Aldrich . Toluene (>99.5%), chloroform (>99.8%), acetone (>99.8%), acetonitrile (>99.8%), and isopropanol (>99.8%) were purchased from Ekos-1 . Deionized water (Milli-Q water) and ethanol  were used as solvents. All chemicals were used as received.CDs samples were obtained by solvothermal heating of the precursors solution. CD-w was synthesized from 0.5 g of o-PD in 25 mL of distilled water. CD-HCl was synthesized similarly to CD-1 with additions of HCl to form acid media (pH = 2). CD-BA-w was synthesized from 0.5 g of o-PD and 0.56 g of benzoic acid dissolved in 25 mL of water. CD-TU-w was synthesized from 0.5 g of o-PD and 0.7 g of thiourea dissolved in 25 mL of water. The amounts of precursors including oxygen for all samples is summarized in Transmission electron microscopy (TEM) images were obtained on a Libra 200FE . Atomic force microscopy (AFM) measurements were carried out using a Solver PRO-M microscope  in the semi-contact mode. For AFM measurements, 10 μL of light-brown CD solutions were spin-casted onto the mica substrates at 2000 rpm for 30 s. XPS measurements were performed on an Escalab 250Xi photoelectron spectrometer with AlKα radiation (photon energy 1486.6 eV). Measurements were performed in the constant pass energy mode at 100 eV for the survey XPS spectrum and at 50 eV for the core level spectra of single elements, using an XPS spot size of 650 μm. Raman spectra were measured on an inVia  microspectrometer equipped with a 20× object and a 514 nm laser source. Fourier-transform infrared (FTIR) spectra were recorded on a Tenzor II infrared spectrophotometer  in an attenuated total reflection mode. Absorption spectra were measured on a spectrophotometer UV-3600 . PL spectra and PL excitation-emission (PLE-PL) maps in the UV-Vis range were collected on a Cary Eclipse . Time-resolved PL measurements were performed on a confocal microscope MicroTime 100  equipped with a 3× objective (NA = 0.1) and a 405 nm pulsed diode laser. PL decay curves were fitted by a biexponential function: 0 = +1.2291 V) [During heating of the precursors mixture in an autoclave, o-PD may undergo an oxidation, the speed and yield of which depend on the amount of oxygen, pH of the solution, and influence of additional precursors. .2291 V) ; (ii) th.2291 V) ; (iii) mAnother way to engineer structural and optical properties of CDs is a heteroatom doping by using additional N,S-containing precursors. For this, we have chosen thiourea (CD-TU sample) which allows to form N,S-doped CDs. In an aqueous solution, thiourea is hydrolyzed with the formation of hydrogen sulfide, which promotes a formation of 2,3-diaminophenotiazin and its subsequent carbonization. Direct reaction between o-PD and thiourea is also possible by the scheme shown in The TEM images a–d show In −1 is attributed to breathing vibrations of benzene rings; the peaks at 1255 and 1380 cm−1 correspond to C-NH bending and C-N stretching [−1 are attributed to phenazine-like structures and 2,3-DAPN [−1, which may indicate a shorter C-N or C=N bond length. Furthermore, the peaks attributed to phenazine-like structures are more pronounced in CD-HCl, CD-BA, and CD-TU samples, thus indicating the formation of a larger amount of phenazine derivatives, as was predicted from chemical reactions in −1, which can be attributed to symmetric, asymmetric vibrations, scissoring of -NH2, and C-N stretching, respectively. Peaks at 3000–3070 and 1540–1600 cm−1 can be attributed to stretching vibrations of C-H and C=C (ring), respectively. Thus, CD-w consists of N-doped aromatic domains with a surface rich in amine groups. For the CD-HCl sample, a peak at 2840 cm−1 with additional peaks at 2500–2600 cm−1 can be attributed to structures similar to imidazole or protonated amines, which agrees well with measurements of the CDs surface charge. These peaks are not intense but are also observed in spectra for both CD-BA and CD-TU. For CD-BA, in addition to peaks observed for CD-HCl, a peak at 1700 cm−1 emerges, which can be attributed to C=O/N-C groups in amides. For CD-TU, a strong peak at 2048 cm−1 typical for N=C=S is observed. Thus, the addition of acids leads to formation of an N-doped phenazine-like carbon network and to larger degree of protonation of amino groups at the CDs surface, whereas addition of thiourea leads to a formation of N, S-codoped aromatic carbon domains within CDs. These observations agree well with the predicted chemical structure of CDs demonstrated in The Raman spectra shown in retching , respect2,3-DAPN , respectIn the absorption spectra shown in 2-domains. It is worth noting that for the CD-w sample, the cooling and heating of the solution slightly affected the optical properties of the CD-w solution , chloroform (0.259), acetone (0.355), acetonitrile (0.46), isopropanol (0.546), ethanol (0.654), and water (1.0). The normalized polarity values  resulted in the absorption peak at 485 nm disappearing, whereas decreasing the polarity of the solvent starting from isopropanol to toluene led to a blue shift of the main absorption band from 460 to 430 nm a. The PLFor CD-TU, the main absorption band was also blue shifted from 440 to 415 nm when the polarity of the solvent was decreased. It should be noted that absorption in the 350–380 nm spectral region increased with a decrease in ENT d. In theENT , it is sThese observations suggest that the optical centers formed in CDs in their excited state have greater polarity than in their ground state, which was also observed for aromatic molecules  and o-PDPL band) . The chaPL band) . It is wPL band) .pH sensing in biological objects including cells and tissues is a current emerging area of applications for colloidal nanoparticles . In thisThe already developed pH sensing probes based on CDs implement change in the PL intensity during the protonation/deprotonation process . It was The PL excitation-emission maps shown in Thus, CD-w sample can be used as pH probe supporting three different methods for pH estimation. This helps expand the pH range of interest and available experimental methods. pH values were calculated from absorption  graph shown in 3+ affected the position of the band and the peak intensity, whereas the presence of Ca2+, Co2+ or Cu2+ led to a decrease in the intensity of the whole band with the maintenance of the O.D.@485/O.D.@460 ratio. The PL band with added Co2+ and Cu2+ maintained the position of the maximum along with a decrease in intensity with increasing concentration of ions  with a varied pH of 4.1 (sample 1), 4.7 (sample 2), and 5.9 (sample 3) were examined. The absorption and PL spectra excited at 360 and 450 nm are shown in PL values, the amount of ethanol in the water can be predicted as 63 and 79% for samples 2 and 3, respectively. For sample 1, since the PL excited at 450 nm was strongly red shifted, the amount of ethanol was estimated by absorption peak position (94%), which shows poor relation to the real value. The pH was estimated by the ratiometric method based on changes in both absorption  spherical particles with sizes up to 15 nm with stable morphology and absorption and PL peak positions under temperature changes in a wide range (20–80 °C) and 6 month storage. The type of additional precursor (acid or thioamine) affects the morphology, including the chemical composition of the surface, which in turn governs the response to media changes. CDs synthesized from o-PD only and with addition of thiourea in water may act as an absorptive/fluorescent sensing probe for solvent polarity with


Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) is an emerging technology that can accurately map cis-regulatory elements in animals and plants. However, the presence of cell walls and chloroplasts in plants hinders the extraction of high-quality nuclei, thereby affects the quality of ATAC-seq data. Meanwhile, it is tricky to perform ATAC-seq with different tissue types, especially for those with limited size and amount. Moreover, with rapid growth of ATAC-seq datasets from plants, powerful and easy-to-use data analysis pipelines for ATAC-seq, especially for wheat is lacking. Here, we provided an all-in-one solution for mapping open chromatin in wheat including both experimental and data analysis procedure. We efficiently obtained nuclei with less cell debris from various wheat tissues. High-quality ATAC-seq data from young spike and ovary, which are hard to harvest were generated. We determined that the saturation sequencing depth of wheat ATAC-seq is about 16 Gb. Particularly, we developed a powerful and easy-to-use online pipeline to analyze the wheat ATAC-seq data and this pipeline can be easily extended to other plant species. The method developed here will facilitate plant regulatory genome study not only for wheat but also for other plant species.Gene transcription is largely regulated by  Cis-regulatory elements, such as promoters, enhancers and insulators recruit transcription factors to form protein-DNA complexes, which regulate the transcription of downstream genes , Deoxyribonuclease I (DNase I)-hypersensitive site sequencing (DNase-seq) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) . The chrArabidopsis and rice  Data Coordinating Center Uniform has proposed a standard ATAC-seq data processing pipeline, which considers alignment rate, library complexity, peak number, insert fragment length, the fraction of reads in called peak regions (FRiP) score and transcription start site (TSS) enrichment to evaluate the data quality . Neverthcis-regulatory elements from the complicated genome in wheat. Currently, there are a few studies to investigate chromatin accessibility in wheat. Moussa Benhamed and colleagues mapped open chromatin in wheat seedlings by ATAC-seq and found that the intergenic condensed spacer (ICONS) border is highly accessible in wheat, while the internal ICONS accessibility is reduced  was used in this study. Seeds were sterilized in 75% alcohol and then washed three times with water. Seeds were germinated on filter paper at 25°C under 16/8 light/dark conditions. Two days later, the radicles were harvested and the remaining part was transferred to the soil. The plants were grown at 22°C in greenhouse. We harvested young spike at double-ridge stage and ovary at 2 days after pollination. Half of the above materials were stored at -80°C for RNA-seq and the other half for nuclei extraction.2, 3 mM DTT, 0.4% Triton X-100 1% BSA, 1 tablet/50 ml protease inhibitor), D-Sorbitol buffer , Chopping Buffer  or PP Buffer . After chopping, the liquid was filtered with 30 μm strainer.Except the double-ridge stage young spike which had been stored at -80°C for more than 1 month, fresh material was collected and was immediately chopped in any of D-S-0.4 buffer  and 1.5 μl TTEMix V50  were added to the nuclei solution and incubated at 37°C for 30 min. The reaction was terminated and DNA was purified using the QIAGEN MinElute PCR Purification Kit . The eluted DNA was used to prepare library with either TruePrep DNA Library Prep Kit V2  or NEBNext High-Fidelity2X PCR Master Mix . The primer sequences are listed in Crude nuclei were stained with DAPI and loaded into a flow cytometer (BD FacsAria II SORP). 50,000 nuclei were sorted into 400 μl buffer. The nuclei were collected by centrifugation at 1,000 g for 10 min at 4°C. After removing the supernatant but 20 μl liquid remained, premixed 1×TD buffer . RNA-seq libraries were sequenced by DNBSEQ-T7  with the 150-bp paired-end mode. RNA-seq was performed in triplicates.Tissue was snap-frozen in liquid nitrogen and stored at -80°C. Total RNA from young spike at double-ridge stage and ovary at 2 days after pollination was extracted using Trizol . The concentration and purity of RNA were detected by Nanodrop (Thermo Fisher Scientific) and electrophoresis. RNA-seq libraries from the two tissues were prepared using the VAHTShttps://github.com/usadellab/Trimmomatic) with parameters of “LEADING: 20 TRAILING: 20 SLIDINGWINDOW: 4: 15 MINLEN: 36”. The clean data was mapped to IWGSCv1.0 using bowtie2 v2.4.4 with parameters “–sensitive -k 3” . In order to remove false positive peaks, we used IDR to get the consistent peaks, which was recommended by ENCODE. The FRiP and the SPOT coefficients were used to evaluate the signal-to-noise ratio of the libraries. The final bam files produced from reads alignment were converted to bigwig format for visualization on genome browser using bamCoverage of deepTools v3.5.0  (https://github.com/hcph/wheatATAC.git).The quality of raw sequencing data was estimated using Trimmomatic v0.32  . We devevia SAMtools toolkit combined with Linux split command and performed peak calling via MACS2  sorts stained nuclei to get rid of other organelles. Combination of FANS with ATAC-seq (FANS-ATAC-seq) reduced the alignment rate of chloroplasts and mitochondria to 30% in bidopsis . To deveHow many reads are needed is always a question for next-generation sequencing based methods. The sequencing depth is quite important for subsequent qualitative and quantitative analysis. Insufficient sequencing data may cause false positive results and lost positive signals due to the interference from background noise. Exceeded sequencing not only wastes money, but also reduces the signal-to-noise ratio. To explore the sequencing saturation of ATAC-seq in wheat, we sequenced one library from each of the young spike and the ovary for 16-17 Gb high quality clean data, which is similar to the size of wheat genome. Then, we randomly extracted from 10% to 200% of the reads from the clean data to call peaks, respectively. The results show that the saturation of sequencing depth is sample dependent, but in general, 16-17 Gb data is optimal for ATAC-seq in wheat Figure 3https://github.com/nboley/idr), respectively  values between original replicates, the pseudo-replicates which respectively and randomly split from each of the original replicate, and the pseudo-replicates that randomly split after pooling the replicates were calculated by IDR v2.0.4.2 (https://github.com/hcph/wheatATAC.git). The users can take raw sequence data as input to obtain OCRs and all the results for quality evaluation . We integrated the quality evaluation process in the pipeline by including the production of visualization files, calculation for library complexity, insert fragment distribution, sample cross correlation, TSS enrichment score, FRiP and the Signal Portion Of Tags (SPOT) values  were treated as intergenic regions where distal regulatory elements like enhancers may exist. The distribution of the predicted OCRs in TSS and TES showed the same trend in young spike and ovary . Two metrics namely FRiP and SPOT values are representative for signal-to-noise ratio of the sequencing data. The FRiP value is calculated based on the number of finally obtained peaks. The SPOT uses the binomial distribution with a local background model to automatically correct for broad-scale regional differences in tag levels; therefore, it is more suggestive than FRiP. However, we recommend using both the FRiP and the SPOT value to evaluate the data quality since FRiP could indicate the efficiency of Tn5 tagmentation. Active transcription is highly correlated with open chromatin, thus chromatin is more likely to be open at the TSS  and the Fundamental Research Funds for the Central Universities (2662020ZKPY002).We sincerely thank the computing platform of the National Key Laboratory of Crop Genetic Improvement in HZAU for providing the computational resources.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.